Protein folding is critical to the survival of all cells; it is the final step in the pathway from DNA to active protein. Proteins use various mechanisms to achieve their final folded state, including spontaneous folding and folding dependent on cellular machines known collectively as chaperones. The class of chaperones called chaperonins assists protein folding in an ATP-dependent manner and is responsible for folding many proteins, both newly synthesized and newly translocated across cellular membranes. They play a role in refolding heat or stress damaged proteins in the recovery from thermal or other insults. In this project, chaperonin-mediated protein folding by the GroEL-GroES system of the extreme thermophile, Thermus thermophilus, will be studied with the long-term goal of understanding how cellular systems have evolved to deal with environmental extremes and, more specifically, what the differences are between folding mechanisms in mesophilic organisms and those in thermophiles. The specific aims of this project are to: 1) identify potential substrate proteins for the thermophilic chaperonin, in addition to the 24 reported recently, by proteomic analysis of the protein occupants of the chaperonin complex recovered from T. thermophilus cells and evaluate both sets of proteins for their dependence on the chaperonin system for refolding in vitro, examining aggregation and chaperonin binding when diluted from denaturant, release by the addition of ATP and co-chaperonin, and recovery of native enzymatic activity or protein function; 2) investigate the ATPase and protein folding cycles of the T. thermophilus chaperonin system and determine the efficiency of refolding in terms of cycles of ATP hydrolysis required to fold a given protein, comparing this to the efficiency of the well-studied E. coli chaperonin system, both with homologous substrate proteins and with identical mesophilic or thermophilic proteins at the same temperature; and 3) initiate NMR studies of a thermophilic substrate protein bound to thermophilic GroEL, with the expectation that the ability to collect spectra at elevated temperature may provide new insights into the nature of the bound protein and its interaction with the chaperonin. Not only is the description of how proteins fold central to our understanding of how cells survive and grow, it is crucial to uncovering the causes behind diseases that result from protein misfolding. These include the amyloidoses, such as Alzheimer disease, prion diseases, such as "mad cow" disease, and certain genetic diseases, such as the most common form of cystic fibrosis. Understanding the processes of protein folding may lead to the uncovering of new drugs and new therapeutic approaches to these severe disorders. Page 2 Number pages consecutively at the bottom throughout Form Page 2 [unreadable] [unreadable] [unreadable]